Bone marrow mesenchymal stem cell-derived exosomal miR-30e-5p ameliorates high-glucose induced renal proximal tubular cell pyroptosis by inhibiting ELAVL1

Abstract Background The rapid increase in the prevalence of diabetes has resulted in more cases of diabetic kidney disease (DKD). Treatment with bone marrow mesenchymal stem cells (BMSCs) may represent an alternative strategy to manage DKD. Methods HK-2 cells were treated with 30 mM high glucose (HG). Bone marrow MSC-derived exosomes (BMSC-exos) were isolated and internalized into HK-2 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays were used to measure viability and cytotoxicity. The secretion of IL-1β and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. Quantitative RT-PCR was used to measure the levels of miR-30e-5p, ELAV like RNA binding protein 1 (ELAVL1), IL-1β, and IL-18. The expression of ELAVL1 and pyroptosis-associated cytokine proteins was determined by western blot analysis. A dual-luciferase reporter gene assay was conducted to confirm the relationship between miR-30e-5p and ELAVL1. Results BMSC-exos decreased LDH, IL-1β, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1β, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion derived from BMSC-exos promoted HK-2 cell pyroptosis. Besides, miR-30e-5p over-expression or ELVAL1 knockdown could directly inhibit pyroptosis. ELAVL1 was a target of miR-30e-5p and knocking down ELAVL1 reversed the effect of miR-30e-5p inhibition in BMSC-exos-treated HK-2 cells. Conclusions BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis by targeting ELAVL1 in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.


Introduction
Diabetic kidney disease (DKD) can cause chronic kidney disease. DKD is associated with albuminuria, progressive loss of kidney function, glomerular hypertrophy, and renal fibrosis [1,2]. The conventional treatment for DKD is effective in the early stages of renal damage, but not at an advanced stage, in which hyperglycemia and hypertension occur [3]. Dialysis and kidney transplantation are also helpful in treating kidney failure, but immune system rejection and a lack of donors are the primary limitation for their overall success [4]. Therefore, it is necessary to identify the mechanism of DKD in detail to develop new treatment strategies.
With the development of stem cell therapy, mesenchymal stem cells (MSCs) have gained interest as a potential therapy for renal damage [5]. MSCs exhibit a self-renewal capacity and multilineage differentiation potential [3]. Bone marrow, adipose tissue, and umbilical cord are the primary sources of MSCs. MSCs have protective effects against diabetic retinopathy, myocardial infarction, diabetes, and DKD [4,[6][7][8]. In addition, MSCs can secrete cytokines, chemokines, growth factors, and extracellular vesicles [9]. Exosomes, which are a type of extracellular vesicle, range in size from 30 to 150 nm and are ubiquitous in body fluid. The underlying mechanism of bone marrow MSC-derived exosomes (BMSC-exos) may involve their function as mediators of intercellular communication and immune regulation [10,11].
Exosomes contain mRNAs, microRNAs (miRNA), and proteins. miRNAs are small noncoding transcripts that can regulate cell proliferation, apoptosis, oxidative stress, inflammatory factors, as well as other functions and participate in the occurrence and development of tumors, diabetes, Parkinson's disease, and other diseases [12]. Recently, the effects of miRNAs on kidney disease have attracted attention. Thus far, miRNA dysregulation was shown to cause DKD, indicating that miRNAs may be useful diagnostic biomarkers for DKD [13]. miR-30e-5p expression was dysregulated in liver carcinoma, chronic myeloid leukemia, lung adenocarcinoma, colorectal cancer, and type 1 diabetes [14][15][16][17]. In addition, miR-30e-5p was predicted to be highly expressed in BMSC-exos; however, it was down-regulated in DKD [18]. Thus, we hypothesized that miR-30e-5p may be effective in regulating HG induced renal tubular epithelial cell injury.
Pyroptosis refers to the process of pro-inflammatory programmed cell death [19]. The NOD-like receptor pyrin containing receptor 3 (NLRP3) inflammasome, ELAV-like RNA binding protein 1 (ELAVL1), and caspase-1 can promote the secretion of inflammatory cytokines and cause plasma membrane rupture, which are considered hallmark features of pyroptosis [20,21]. Moreover, ELAVL1 could be targeted by miR-23c to regulate HG induced renal tubular epithelial pyroptosis [22]. HuR, a member of the ELAV protein family, contributes to ischemia, renal fibrosis, matrix protein accumulation, and angiogenesis. Furthermore, HuR was found to be up-regulated in DKD patients; thus, HuR may play an important role in the pathogenesis of DKD [23]. It was proved that pyroptosis participated in the process of DKD and NLRP3 inflammasomes promoted DKD progression [24,25]. The starBase database (http:// starbase.sysu.edu.cn) predicted that ELAVL1 may be a downstream target gene of miR-30e-5p, but such a relationship has not yet been reported.
In the present study, we hypothesized that bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-30e-5p ameliorates HG-induced HK-2 cell pyroptosis by targeting ELAVL1. Our findings may provide a new strategy for the treatment of DKD.

Cell culture and drug treatment
HK-2 (human renal proximal tubular cells) cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 media containing 10% FBS, hydrocortisone (5 ng/ml), glutamine (2 mM), sodium selenite (5 ng/ml), insulin (5 lg/ml), transferrin (5 lg/ml), penicillin (100 U/ml), and streptomycin (100 lg/ml) in 5% CO 2 /95% humidified air at 37 C. To simulate a DKD model in vitro, 30 mM glucose was added for 72 h to establish the HG model, whereas 5.5 mM glucose was added as a normal (NG) control and an additional 24.5 mM mannitol was added as an isotonic control [17].

Preparation and characterization of BMSCexos
BMSC-exos were obtained from the supernatant of human-BMSCs and collected at a density of 1.0 Â 10 5 cells/well in a six-well plate. They were centrifuged at 2000Âg for 20 min. The conditioned medium was ultracentrifuged at 100,000Âg. Exosomes were washed once with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, St. Louis, MO) and the above procedure was repeated. The exosomes were resuspended in PBS, filtered, and stored at À80 C. Transmission electron microscopy was used to detect the presence of BMSC-exos and the particle sizes were assessed by nano-particle tracking analysis (NTA). The surface markers CD73, CD90, CD105, CD34, CD14, and CD45 were detected by Fluorescence-activated Cell Sorting (FACS) analysis. The surface markers CD63 and TSG101 were also analyzed by western blot analysis. Exosomes (100 lg/ml) were tagged with PKH67, a fluorescent linker dye (Sigma-Aldrich, St. Louis, MO), to trace them within cells. Finally, 30 lg BMSC-exos were added to HK-2 cells to determine their effect [26].

Internalization of BMSC-exos in HK-2 cells
BMSC-exos were labeled with the PHK67 membrane dye and 100 lg/ml were added to 3 Â 10 4 HK-2. The cells were then washed with PBS and fixed with paraformaldehyde. The nucleus was counterstained with DAPI (1:200) and observed by fluorescence microscopy (Nikon, Tokyo, Japan) [27]. Cells were seeded into 96-well plates at a density of 2000 cells per well. After a 24-h incubation, the medium was removed and MTT (5 mg/ml, M2003, Sigma-Aldrich, St. Louis, MO) was added to each well. After 3 h, the MTT was removed and 200 ll of DMSO were added to each well. The absorbance was measured at 490 nm using a microplate reader (Tecan, Mannedorf, Switzerland).

Flow cytometry
HK-2 cells were plated in a six-well plate at a density of 2 Â 10 5 cells per well. After 48 h, the cells were harvested, washed with PBS, and centrifuged at 1500 rpm for 5 min. FAM-YVAD-FMK (FAM600-1, Cell Technology, Hayward, CA), caspase-1 antibody, and propidium iodide were added to the resuspended cells and mixed. After incubating at room temperature in the dark for 1 h, the cells were analyzed using a BD Biosciences FACS Calibur system (San Jose, CA).

Measurement of lactate dehydrogenase (LDH) level
Cells (1 Â 10 6 ) were seeded into six-well cell culture plates for 12 h and after treatment, the cell supernatant was collected. LDH levels were measured using an LDH assay kit (C0016, Beyotime, Shanghai, China) based on the manufacturer's instructions. The absorbance was detected at 490 nm using a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA).

qRT-PCR analysis
Total RNA was isolated using Trizol reagent (15596018, Ambion, Austin, TX) and stored at À80 C for further detection. For mRNA analysis, cDNA was synthesized using the SuperScript TM IV First-Strand Synthesis System (6210A, Takara, Otsu, Japan), and qPCR was done using SYBR Green PCR Mastermix (SR1110, Solarbio, Beijing, China). For miRNA analysis, cDNA was synthesized using the Hairpin-it TM Real-Time PCR kit (E22001, GenePharma, Shanghai, China), which was analyzed using the corresponding U6 snRNA Normalization RT-qPCR Quantification Kit (E20001, GenePharma, Shanghai, China). GADPH was served as internal reference for mRNA analysis and U6 served as internal reference for miRNA analysis. Data were analyzed using by the 2 À᭝᭝Ct method. The primer sequences are listed in Table 1.

Western blot analysis
Total protein extracts from HK-2 cells following various treatments were prepared in RIPA buffer containing protease inhibitors. The protein concentration was measured using a BCA assay kit (P0009, Beyotime, Shanghai, China). Thirty micrograms protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Cytiva, Boston, MA). The membranes were blocked in TBS buffer containing 5% skim milk for 1 h and incubated with primary antibodies at 4 C overnight. The membranes were washed with TBST and incubated with the corresponding secondary antibody for 1 h. The signal was detected using an ECL Kit (Thermo

Statistical analysis
All experiments were conducted at least three times and the data were presented as the means ± standard deviations. The data were analyzed using a one-way ANOVA followed by the least significance difference test. A p value <.05 was considered statistically significant.

Isolation, identification, and internalization of BMSC-exos
The primary morphology of exosomes was ellipsoidal or cup-shaped as determined by transmission electron microscopy ( Figure 1(A)). The size was measured by NTA and the diameter was ranged from 30 to 150 nm (Figure 1(B)). As shown in Figure 1(C), the positive markers, CD63 and TSG101 could be detected on BMSC-exos, whereas the negative marker calnexin was not detected in a western blot assay. Flow cytometry revealed that the exosomes were positive for CD73 (99.74%), CD90 (99.41%), and CD105 (99.79%), but negative for CD34 (0.11%), CD14 (0.16%), and CD45 (0.04%) (Figure 1(D)). The fluorescent dye PKH67 could stably bind to the lipid region of membranes and emit green fluorescence, thus enabling exosomes to be traced after co-culturing with cells. As shown in Figure  1(E), the marker protein PKH67, which was internalized into HK-2 cells, was successfully traced in the co-culture medium of BMSC-exos and HK-2 cells, indicating that BMSC-exos were absorbed by HK-2 cells. In summary, BMSC-exos were isolated, identified, and absorbed and internalized by HK-2 cells.

Effect of BMSC-exos on pyroptosis induced by HG
The role of BMSC-exos in pyroptosis induced by HG was evaluated. Cell viability was notably decreased in the HG group, but the inhibitory effect was reversed by BMSC-exos (Figure 2(A)). BMSC-exo treatment markedly decreased secreted LDH, IL-1b, and IL-18 levels in HGtreated cells (Figure 2(B,C)). MiR-30e-5p was highly expressed in the NG, MA, and HG þ Exos groups, but the expression of miR-30e-5p in the HG group was decreased (Figure 2(D)). As shown in Figure 2(E,F), the level of pyroptosis was markedly decreased after BMSCexos treatment in HG-induced cells. The expression of IL-1b and IL-18 mRNA in the HG group was up-regulated compared with the NG and MA groups, whereas they were significantly decreased in the HG þ Exos group (Figure 2(G)). In addition, Figure 2(H,I) shows that the pyroptosis markers, GSDMD-N, IL-1b, NLRP3, and caspase-1, were markedly increased following HG treatment, but significantly decreased after BMSC-exo treatment. These results indicate that BMSC-exos ameliorate pyroptosis.

Discussion
Both type I and type II diabetes can induce DKD [28]. However, despite extensive efforts in disease control, many patients develop not only early-and middle-stage DKD, but also end-stage chronic kidney disease. The current treatments remain limited and novel therapeutics are needed to control the progression of this disease. In the present study, BMSC-exos were shown to alleviate HG-induced HK-2 cell pyroptosis. Furthermore, BMSC-derived exosomal miR-30e-5p ameliorated HK-2 cell pyroptosis by modulating ELAVL1.
In the past few decades, MSCs have gained interest as a potential therapy for DKD. MSCs have many physiological functions including self-renewal, clone cell population generation, and multi-line differentiation [29]. As a treatment for DKD, MSCs were shown to reduce hyperglycemia and proteinuria, as well as improve renal pathological changes [30][31][32]. BMSC-exos can serve as vehicles to treat DKD based on their ability to exchange bioactive components and transport genetic contents [33]. In the present study, we demonstrated that BMSC-exos ameliorate pyroptosis in HGinduced HK-2 cells. Our findings provided an alternative strategy for the treatment of DKD, although the putative mechanism needs clarification.
Several studies have shown that various miRNAs are associated with DKD [31,32]. For example, miR-185 was reported to regulate the deposition of the DKD extracellular matrix by targeting TGF-b1 [34]. The expression of miR-497 was down-regulated in the kidneys of mice with diabetes [35]. As an emerging regulator of disease progression, exosomal miRNAs are associated with a class of metabolic disorders, including diabetes and its complications [22]. Exosomal miR-215-5p derived from adipose-derived stem cells was protective against migration and inhibited podocyte damage in DKD [22]. Furthermore, exosomal miR-19b-3p derived from tubular epithelial cells inhibited inflammation to regulate DKD progression [23]. To our knowledge, miR-30e-5p has an important role in diabetes. We demonstrated the beneficial effect of BMSC-exos on HG-induced HK-2 cell pyroptosis by transferring miR-30e-5p. This may also be helpful for using miR-30e-5p to regulate DKD.
Recent evidence has indicated that pyroptosis, a proinflammatory mode of programmed cell death, is involved in DKD [36]. Due to chronic exposure to diabetic substrates, pyroptosis and renal inflammation occur [37]. During the progression of pyroptosis, caspase-1 is activated by a large supramolecular complex known as the pyroptosome, which subsequently activates IL-1b and IL-18. In the present study, HG acted as a pyroptosis inducer. LDH, IL-1b, and IL-18 secretion was increased in the HG group as well as GSDMD-N, IL-1b, NLRP3, and caspase-1 expression. ELAVL1, a component of the pyroptosome involved in activating pyroptosis, promotes IL-1b and IL-18 secretion combined with caspase-1 activation [22]. Another important finding of this study was the binding relationship between ELAVL1 and miR-30e-5p. We found that miR-30e-5p could inhibit caspase-1-mediated pyroptosis in HG-induced HK-2 cells by targeting ELAVL1.
The nephron is the basic unit of kidney structure, including the renal body and connected renal tubules, which play an important role in regulating kidney function. The abnormal function of renal tubular epithelial cells is a key factor leading to DKD. Apoptosis of renal tubular epithelial cells results in abnormal renal tubular re-absorption and renal fibrosis [38]. HK-2 cells have most of the functional characteristics of normal adult renal proximal tubular cells, thus they are widely used to study renal pathology and screen therapeutic drugs in vitro [39]. This study focused on the regulatory mechanism of BMSC-exo-derived miR-30e-5p on HG-induced pyroptosis in HK-2 cells. The main pathological features of DKD include glomerular hypertrophy, increased production and accumulation of extracellular matrix, mesangial expansion, basement membrane thickening, glomerular follicular loss, and renal fibrosis, which leads to the progressive loss of renal function [40]. Whether miR-30e-5p affects these pathological changes in DKD in vivo needs to be confirmed. In addition, the glomerular filtration barrier consists of the basement membrane, the inner endothelium, and the outer podocyte epithelium. DKD was shown to be associated with necrosis of podocytes and mesangial cells [41,42]. Moreover, high glucose (HG) levels inhibited autophagy by activating the JAK/STAT pathway of podocytes, thereby preventing the effective removal of damaged proteins and organelles from the body to prevent apoptosis, and ultimately, aggravating podocyte injury and DKD progression [43]. Glomerular endothelial cell injury is thought to contribute to the development of microalbuminuria, an early event in DKD [44]. In addition, recent studies suggested that glomerular endothelial cells may contribute to DKD through paracrine communication with other glomerular cells, such as podocytes and mesangial cells. Therefore, in addition to tubular epithelial cells, mesangial cells, and podocytes also play an important role in apoptosis, necrosis, and autophagy in DKD. However, whether BMSC-exoderived miR-30e-5p regulates apoptosis, pyroptosis, or the necrotizing apoptosis process of other renal cells or downstream signaling warrants further investigation.
In conclusion, our findings demonstrate the ameliorative effect of BMSC-derived exosomal miR-30e-5p on HG-induced HK-2 cell pyroptosis. The underlying mechanism involves the inhibitory effect of miR-30e-5p on ELAVL1 and caspase-1-mediated pyroptosis. However, our work lacks clinical and animal experiments for further validation. In subsequent studies, we will certainly explore the effects of BMSCs-exos on the development of DKD in vivo. Our study is expected to provide new target and direction for the therapy of DKD.